Additional characterization of a hexanucleotide polymorphic site in the first intron of human H-ras gene: comparative study of its alterations in non-small cell lung carcinomas and sporadic invasive breast carcinomas.

ABSTRACT

Intron 1 of the human H-ras gene possesses a polymorphism consisting of repetitions of the GGGCCT consensus. Three alleles have been reported at this locus. We confirmed that two, P1 and P2, display four and two repeats, respectively, with their internal sequence structure similar to that previously described. The third, P3, previously assigned as a three-unit repetition allele according to its electrophoretic mobility and with no other information regarding its internal structure, was also found. Sequence analysis of the P3 allele revealed that it consists of three perfect repeats of the GGGCCT consensus. This polymorphism is present only in human c-H-ras gene, although single hexanucleotide repeats are found scattered within intron 1 of this gene in rodents. Analysis of this locus in matched tumor/distant normal samples from (i) 38 patients with non-small-cell lung carcinoma (NSCLC), and (ii) 35 patients with sporadic invasive breast carcinoma, revealed (1) 6.6% and 19% loss of heterozygosity (LOH) respectively, and (2) 10.5% and 2.9% hexanucleotide instability (HI) respectively, detected by the presence of shifted in length alleles. Shifted alleles exhibited altered internal sequence structure in comparison to normal ones, suggesting complex mutational events. The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias adjacent to squamous cell carcinomas (7.7% LOH, 5.9% HI), implying that abnormalities at this locus may be early events in lung carcinogenesis. The frequency of alterations (LOH vs. HI) was significantly different among NSCLC and breast cancer (P=.005), probably due to the different tumor biology of each system. Finally, altered mRNA expression of H-ras gene was detected in all cases with HI, but this finding was also observed in samples without HI. In view of reports showing that elements in intron 1 of H-ras gene potentially influence its transcriptional regulation, from our results we cannot exclude that the hexanucleotide locus could be an element with possible involvement in expressional regulation of this gene.